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      c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress

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          Abstract

          The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP ( GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has di adenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13–22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.

          Author Summary

          Staphylococcus aureus is an important human pathogen that colonizes the nares and skin of both sick and healthy individuals and causes a variety of infections ranging from superficial skin to invasive infections. The ability of this bacterium to cause disease depends on many factors and is, in part, due to multi-functional cell surface structures. One such structure is lipoteichoic acid (LTA), which is crucial for bacterial growth. In this study we show that LTA is also important for growth of a clinically relevant community-acquired methicillin resistant S. aureus (CA-MRSA) strain and not only for laboratory strains as previously described. We set out to investigate if S. aureus can find a way to survive without LTA and identified strains that can grow and divide almost normally in its absence. Using a whole genome sequencing approach, we found that alterations in one gene, gdpP, allow these strains to grow in the absence of LTA. We show that this mutation causes an increase in the recently identified signaling molecule, c-di-AMP, within the cell. Therefore, with this study we provide information on one of the first functions of this novel secondary messenger, which is in helping bacteria to cope with extreme cell wall stress.

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          Most cited references52

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          Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan.

          Although the role of Toll-like receptors in extracellular bacterial sensing has been investigated intensively, intracellular detection of bacteria through Nod molecules remains largely uncharacterized. Here, we show that human Nod1 specifically detects a unique diaminopimelate-containing N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) tripeptide motif found in Gram-negative bacterial peptidoglycan, resulting in activation of the transcription factor NF-kappaB pathway. Moreover, we show that in epithelial cells (which represent the first line of defense against invasive pathogens), Nod1is indispensable for intracellular Gram-negative bacterial sensing.
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            c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response.

            Intracellular bacterial pathogens, such as Listeria monocytogenes, are detected in the cytosol of host immune cells. Induction of this host response is often dependent on microbial secretion systems and, in L. monocytogenes, is dependent on multidrug efflux pumps (MDRs). Using L. monocytogenes mutants that overexpressed MDRs, we identified cyclic diadenosine monophosphate (c-di-AMP) as a secreted molecule able to trigger the cytosolic host response. Overexpression of the di-adenylate cyclase, dacA (lmo2120), resulted in elevated levels of the host response during infection. c-di-AMP thus represents a putative bacterial secondary signaling molecule that triggers a cytosolic pathway of innate immunity and is predicted to be present in a wide variety of bacteria and archea.
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              Essential genes of a minimal bacterium.

              Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metabolism and little genomic redundancy. Consequently, its genome is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. Using global transposon mutagenesis, we isolated and characterized gene disruption mutants for 100 different nonessential protein-coding genes. None of the 43 RNA-coding genes were disrupted. Herein, we identify 382 of the 482 M. genitalium protein-coding genes as essential, plus five sets of disrupted genes that encode proteins with potentially redundant essential functions, such as phosphate transport. Genes encoding proteins of unknown function constitute 28% of the essential protein-coding genes set. Disruption of some genes accelerated M. genitalium growth.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                September 2011
                September 2011
                1 September 2011
                : 7
                : 9
                : e1002217
                Affiliations
                [1 ]Section of Microbiology, Imperial College London, London, United Kingdom
                [2 ]Department of Life Sciences, Imperial College London, London, United Kingdom
                [3 ]Institute of Pharmacology, Hannover Medical School, Hannover, Germany
                Dartmouth Medical School, United States of America
                Author notes

                Conceived and designed the experiments: RMC AG. Performed the experiments: RMC HB AG. Analyzed the data: RMC JCA VK AG. Contributed reagents/materials/analysis tools: RMC JCA VK AG. Wrote the paper: RMC AG.

                Article
                PPATHOGENS-D-11-00501
                10.1371/journal.ppat.1002217
                3164647
                21909268
                173a1cba-343c-4511-9be4-a30559fd35b8
                Corrigan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 11 March 2011
                : 8 July 2011
                Page count
                Pages: 16
                Categories
                Research Article
                Biology
                Biochemistry
                Immunology
                Microbiology
                Medicine
                Infectious Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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